A series of designed peptide 33mers (βpep peptides) has been reported to be bactericidal and to be capable of neutralizing the bacterial endotoxin lipopolysaccharide (LPS) (Mayo et al., Biochim. Biophys. Acta 1425, 81–92 (1998)). CD and NMR conformational analyses indicate that βpep peptides form β-sheets (Mayo et al., Protein Sci. 5, 1301–1315 (1996)), and one of these, βpep-4, folds compactly as anti-parallel β-sheet sandwich (Ilyina et al., Biochemistry 36, 5245–5250 (1997)). βpep-19 is potently bactericidal in the 10−8 M range (Mayo et al., Biochim. Biophys. Acta 1425, 81–92 (1998)). βpep peptides function like Limulus anti-LPS factor (LALF) and the homologous bactericidal/permeability increasing protein (B/PI) (Hoess et al., EMBO J. 12, 3351–3356 (1993)) in that all appear to express activity through an amphipathic β-sheet structural motif having a cationic β-sheet face (Kelly et al., Surgery 114, 140–146 (1993); Siefferman et al., Infect. Immun. 59, 2152–2157 (1991); Beamer et al., Science 276, 1861–1864 (1997); and Gray et al., Biochim. Biophys. Acta 1244, 185–190 (1995)).
Numerous studies on bactericidal peptides indicate the functional importance of a net positive charge and high hydrophobicity in the context of an amphipathic, usually helical, structure (Maloy et al., Biopolymers 37, 105–122 (1995)). The net positive charge promotes interaction with the negatively charged surface of bacterial membranes (Matsuzaki et al., Biochemistry 36, 2104–2111(1997)), whereas structure-activity relationships demonstrate that the amphipathic conformation of the peptide promotes bacterial cell lysis (Andreu et al., Biochemistry 24, 1683–1688 (1985)). Bactericidal peptides such as cecropins (Lee et al., Proc. Natl. Acad. Sci. USA 86, 9159–9162 (1989)), magainins (Zasloff, Proc. Natl. Acad. Sci. USA, 84, 5449–5453 (1987)), proline-arginine-rich peptides (Agerberth et al., Eur. J. Biochem. 202, 849–854 (1991)), and sapecin (Matsuyama et al., J. Biol. Chem. 263, 17112–17116 (1988)), like βpep peptides, all have a net positive charge and considerable hydrophobic character. The cecropins and magainins are helix-forming peptides (Holak et al., Biochemistry 27, 7620–7629 (1988); and Marion et al., FEBs Lett. 227, 21–26 (1988)), whereas the sapacins contain both α-helix and β-sheet segments (Hanzawa et al., FEBs Lett. 269, 413–420 (1990)). Structures for the proline-arginine-rich peptides are unknown. Tachyplesin, a bactericidal and endotoxin neutralizing peptide isolated from hemocytes of the horseshoe crab (Kawano et al., J. Bio. Chem. 265, 15365–15367 (1990)), as well as anti-bacterial peptide defensins (Selsted et al., J Biol. Chem. 264, 4003–4007 (1989); and Lebrer et al., Cell 64, 229–230 (1991)), form dimeric β-sheets which are stabilized by three intramolecular disulfide bridges (Hill et al., Science 251, 1481–1485 (1991)). In addition, a number of small, antibiotic peptides based on the structure of the anti-LPS, cyclic peptide polymyxin B (Morrison et al., Immunochem. 13, 813–818 (1976)) have been designed as short β-hairpins constrained by a disulfide bridge (Rustici et al., Science 259, 361–365 (1993)).